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Identification of Oogonial Stem Cells in Adult Quail Ovaries: a Model for Conseryation Research of Endangered a Vian Species

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Identification of Oogonial Stem Cells in Adult Quail Ovaries: a Model for Conseryation Research of Endangered a Vian Species CALIFORNIA STATE UNIVERSITY SAN MARCOS PROJECT SIGNATURE PAGE PROJECT SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENTCE IN BIOTECHNOLOGY PROJECT TITLE: IDENTIFICATION OF OOGONIAL STEM CELLS IN ADULT QUAIL OVARIES: A MODEL FOR CONSERYATION RESEARCH OF ENDANGERED A VIAN SPECIES AUTHOR: SARA JABALAMELI DATE OF SUCCESSFUL DEFENSE: 4/20/2017 THE PROJECT HAS BEEN ACCEPTED BY THE PROJECT COMMITTEE IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER SCIENCE IN BIOTECHNOLOGY DR. BETSY READ 8=,t-~~d r;;; /9 /ao )7 PROJECT COMMITTEE CHAIR SIGNAT DATE > DR. THOMAS SPADY ~-r- 1-t PROJECT COMMITTEE MEMBER DATE EXECUTIVE SUMMARY Identification of oogonial stem cells in adult quail ovaries: A model for conservation research of endangered avian species San Diego Zoo Institute for Conservation Research Sara Jabalameli May 2017 Professional Science Masters in Biotechnology California State University San Marcos The dogma in reproductive physiology today is that higher vertebrates are born with a fixed quantity of oocytes, as the oogonia lose their mitotic ability and initiate maturation after birth or hatch. Males have the ability to maintain a constant pool of spermatozoa via mitotic division of germ-line stem cells known as type A dark spermatogonia. These type A dark spermatogonia in adult male avian species continually go through mitosis to keep up with the lifelong demand of spermatogenesis. The presence of spermatogonial stem cells within adult male testes could make it possible to isolate the stem-cells from the gonads of deceased male birds followed by xenotransfer to domestic host embryos. Depending on the environmental conditions, it is possible to isolate and use germline stem cells within hours to days following a bird’s death. This technique can, if successful, have a significant effect on the conservation of valuable endangered species, as well as maintaining genetically diverse captive populations. Although the xenotransfer of adult germ-line stem cells from deceased bird testes to host chicken embryos is a promising method for saving the germplasm, no study to date has provided the evidence for the existence of oogonial stem cells within adult bird ovaries. In this study, total RNA from adult quail ovaries was isolated and the presence of oogonial stem cells was evaluated by filtering dispersed ovarian stromal cells and performing quantitative PCR using primers for stem cell genes and the germline specific genes 1) Deleted in Azoospermia Like (DAZL), 2) Vasa homolog (VH), and 3) the meiosis specific gene Synaptonemal Complex 3 (SCP3). All three germline specific genes as well as well as stem cell genes were detected, suggesting the presence of oogonial stem cells and/or unorganized germ cells within adult quail ovarian stroma. The presence of oogonial stem cells within adult quail ovaries was quantitatively evaluated using fluorescein isothiocyanate (FITC)-conjugated SSEA-1, SSEA-3, and SSEA-4 antibodies and flow cytometry. Previous studies confirm that SSEA-1, SSEA-3, and SSEA-4 are markers for avian embryonic primordial germ cells (PGCs). The oogonial and spermatogonial stem cells isolated from deceased individuals of endangered bird species could be used to produce donor-derived sperm and eggs by host hens and roosters, significantly impacting future avian conservation. SAN DIEGO ZOO INSTITUTE FOR CONSERVATION Cdliforn~a. State Uniiversity RESEARCH, SAN MARCOS Identification of oogonial stem cells in adult quail ovaries: A model for conservation research of endangered avian species Sara Jabalameli Biotechnology Professional Science Masters California State University, San Marcos San Diego Zoo Institute for Conservation Research Outline: Introduction Hypothesis Objectives Specific Aims 1. qPCR 2. Flow cytometry Results and conclusions Project conclusions Impact Next steps References Introduction: Need for new avian conservation techniques As the number of endangered and critically endangered species increases, the need for new, innovative and practical conservation techniques becomes indispensable BIRDS SEEN STEPPE IN NCR EAGLE 8 INDIAN SPECIES HAVE BEE ADDED TO THE IUC RED LIST Common pochard NEAR-THREATENED CURL W DPIPER • s eppe • orthem lapwing • Red knot eagle • Curle sandpiper • Eurasian Northern oystercatcher • Bar-tailed god it lap ing Curlew sandpiper VULNERABLE • Horned grebe • Common poc hard ENDANGERED Helmeted Hornbill is critically • Steppe eagle endangered IUCN Red List 2016 Introduction: Risks associated with isolating embryonic germ cells The use of PGCs and GGC from endangered exotic embryos is an impractical method due to imposed risks associated with losing the embryo Introduction: Practicality of use of adult germline stem cells The isolation of germline stem cells from deceased adult avian gonads and their xentotransfer into early host chicken embryos is a practical method Stage 15, 50-55 hours Spermatogonial stem cells within A practical Guide to avian deceased adult quail testis Embryonic development Jensen et al. Introduction: Origin and migration of PGCs in avian embryos The xenotransfer of adult stem cells from a donor to a domestic host must occur when the endogenous primordial germ cells start migrating to the gonadal ridge .> ·=··., . Germinal Entrance into Colonization of Area Pellucida Transfer by extra- and intro- embryonic blood stream crescent Extra-embryonic gonadal anlagen Blood vessels Successful xenotransfer Origin and migration of PGCs within avian embryo gonads Introduction: Dogma about the presence of OSCs in adult ovaries Few studies show the success of xenotransfer and colonization of SSC within host embryos but to date no study has proven the existence of oogonial stem cells in adult avian ovaries From Primordi~I Germ Cells @5 '----@ Divisions / @5 '@, ~\ .sr Some cells of -th• \~-:(® - \:) • oOgonial . = 1/" l ino ® Many Follic less -h1ch s +art to develop foil -to reach the -final growth s-tege Such -fol1 iclea eventally de.Keneroi-•- T ermina l p e rio d of rapid gro""i-h Secondary sex cords and accumulotion of Deutoplasm Primary Liberated Oocyte from ~ Primary 05cyte in First Maturation Division Second~ry 05cyte in Second Maturation Division Second \ Polocyte ....., .,. .__ ~ Nonfunctional Ootids Functionol Ootid or Organized germ cells Motured Ovum The early embryology of the Jensen, T, 1999 chick. B Patten, Third edition Hypothesis: Oogonial stem cells exist within adult avian ovaries based on previous studies in mammals and the presence of oogonial stem cell within mammalian ovaries Evidence for an Oogonial STEMCELL Stem Cell Advancing Sttm Ctll Scltnct nature .__ _,,,,, meaicine --~- Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age Human ovarian cortex dissociated tiss women. White et.al. Till½ 2012 The quest for human ovarian stem cells FACS sor=iing AntibodY, ,A DDX4 Telfer and Albertini, 2012 (figure below) So atic cells G> G) Mitotically active OSCs G FP-positive oocyte I Transfected in primordial follicles +with GFP / _ / 7- 14days GFP expressing OSCs Xenograft Evidence for the presence of oogonial stem cells within adult mammalian ovaries Objectives The objective of this study is to determine whether oogonial stem cells exist within adult avian ovaries Jensen, T, 1999 Specific aims: The specific aims of this study are to determine the presence of oogonial stem cells within adult ovaries using two independent methods of 1. qPCR and 2. flow cytometry Gene qVH qDAZL qCSF-1 qPLZF qPIW'IL-2 qC-Kit q0ot4 qSCP3 QuantStudio 6 qPCR machine qB-aot:in BD FACSCalibur flow cytometer Results: Amplification plots: PLZF representative graph (Specific Aim 1) Results show that all analyzed genes were present in adult male and female gonads while the gDNA in adult male testis was not amplified in any of the sample for any of the genes Amplification plot for PLZF gene within Female 3 stroma: Plot 10.0 1.0 c: 0.1 er 0.01 0.001 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Results: melting curve plots: PLZF representative graph (Specific Aim 1) Results show that there was a single sharp peak for all amplified products, while none of the gDNA samples were amplified by primers Melting curve plot for PLZF gene within Female 3 Stroma: Curve Plot 3.0 2.5 C -a:: ...!,.. ~ QJ 2.0 t:: 0 a. QJ ~ QJ 1.5 > +J IV > ·.: 1.0 QJ C) 0.5 67.0 72.0 77.0 82.0 87.0 92.0 Temperature (·C) • cmry stroma adul • T adu Results: Presence or absence of studied genes in studied tissues (Specific Aim 1) Except for germline specific genes qVH, qDAZL, and qSCP3, all genes were present in all analyzed tissues. qVH, qDAZL, and qSCP3 genes were absent in the heart and liver tissues as predicted qPCR results Female1 Stroma Female2 stroma Female3 Stroma Liver Heart qVH + + + − − qDAZL + + + − − qCSF-1 + + + + + qPLZF + + + + + qPIWIL + + + + + qcKit + + + + + qSCP3 + + + − − qB-actin + + + + + qOct4 + + + + + Specific Aim 1 conclusions: qPCR analysis from 3 adult ovarian stroma in the study confirmed the presence of germline specific genes qVH, qDAZL, and qSCP3. However, the presence of SCP3 in the ovary suggests 4 different possibilities 3 possible explanations for the presence of SCP3 in analyzed cells: 1. Cells escaped through the filter and the positive signal is from primordial follicles 2. OSCs are present within the adult ovary, and the positive SCP3 is due to cells transcribing the gene for preparation for differentiation and meiotic division 3. There are still remnants of the secondary sex cords and there are unorganized germ cells arrested at diplotene of prophase of meiosis I within the ovarian stroma 4. cDNA contamination from adult quail testis within the reactions? Flow Cytometry and Analysis results (Specific Aim 2) Flow cytometry was used as an independent method to confirm the presence of OSC in adult quail ovaries by using a BD FACSCalibur™ flow cytometer and FlowJo software No Antibody control {False Positive) SSEA-1 (lmmunopositve) ,.. ~-------------------~ ,.. ~-------------------~ FSC/SSC of No Ab control (10.1% selected) ... ... 1000 - -.,..-- -,,-,,~_. _- - --~,--..~-~- -.-­ ..
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